Development of a liver-specific Tet-off AAV8 vector for improved safety of insulin gene therapy for diabetes.

Background: Diabetes mellitus is caused by a partial or complete lack of insulin production in the body. We have previously shown that a single injection of an adeno-associated virus serotype 8 (AAV8) vector carrying a modified and codon optimized human insulin gene induced hepatic production of insulin and corrected streptozotocin (STZ)-induced diabetes in mice for more than 1 year. Insulin production was constitutive, analogous to long-acting insulin therapy.

Methods: We have developed a single AAV8 vector with a Tet-Off regulatable system as a safety mechanism to turn off insulin secretion should hypoglycaemia develop in vector-treated diabetic mice. We first transfected HepG2 cells or freshly isolated rat hepatocytes in vitro with the Tet-Off system (pAAV-Tetoff -Alb-luc) regulating a luciferase reporter gene. We subsequently incorporated a furin-cleavable codon-optimised human proinsulin cDNA into pAAV-Tetoff backbone to form the doxycycline inducible pAAV-Tetoff -Alb-hINSco.

Results: Using STZ-induced diabetic mice, we were able to switch off insulin secretion repeatedly with doxycycline administration, and showed full restoration of insulin secretion on withdrawing doxycycline.

Conclusions: The present study provides proof of concept that, under circumstances when inappropriate basal insulin secretion is a safety concern, insulin secretion from AAV8 gene therapy can be turned off reversibly with doxycycline.